The β-trefoil protein human fibroblast growth factor-1 (FGF-1) is made up of a six-stranded anti-parallel β-barrel closed off on one end by three β-hairpins, thus exhibiting a three-fold axis of structural symmetry. The N- and C-termini β-strands hydrogen bond to each other and are postulated from both NMR and X-ray structure data to represent a structurally-weakened region of the β-barrel. Val mutations within the N- and C-termini β-strands are shown to stabilize the structure and to increase van der Waals contacts by filling local cavities present within this region. Mutations that increase van der Waals contacts between both the N- and C-termini β-strands are associated with significant reductions in the unfolding kinetics, and also increase the cooperativity of unfolding. These results indicate that an important step in the unfolding of FGF-1 involves the melting of the N- and C-termini. A series of stabilizing mutations are subsequently combined and result in a doubling of the ΔG of unfolding. These mutations simultaneously introduce a three-fold symmetric constraint upon the primary structure. The results support the hypothesis that a symmetric primary structure within a symmetric superfold is a solution to achieve a foldable polypeptide. The results also suggest that the β-trefoil is capable of substantial thermal stability. When considering the “function/stability trade-off” hypothesis, the β-trefoil architecture therefore appears capable of diverse functional adaptation.
Accordingly, mutants of human fibroblast growth factor 1 (FGF1) are described that have enhanced stability and mitogenic potency. In comparison to wild-type FGF1, polypeptides having mutations at positions 12 and 134 exhibit enhanced properties of stability and/or mitogenic activity. Enhanced stability may preclude the need for added heparin in formulations of FGF1 for therapeutic use. Additionally, the enhanced thermal stability may translate to a longer shelf-life and minimization of aggregation during storage. The enhanced mitogenicity, which is possibly related to enhanced stability, may provide for use of smaller dosages for equivalent efficacy.